The present invention relates to a novel enzyme which is useful for an optical resolution of D,L-pantolactone through a D-selective asymmetric hydrolysis process and also to a gene encoding the same. More particularly, the present invention relates to proteins having a natural D-pantolactone hydrolase activity, produced by Fusarium oxysporum, or an activity substantially equivalent to the same and genes coding for the same. Specifically, the present invention relates to DNA containing a nucleotide sequence coding for said protein; to host cells transformed or transfected with said DNA; to a process for the production of said D-pantolactone hydrolase protein via using said host cells; and to the use of such proteins and host cells.
D-Pantolactone has been known as an intermediate in the preparation of D-pantothenic acid and pantethine which are useful as vitamins of medical or physiological importance. D-Pantolactone has heretofore been prepared through an optical resolution of a chemically-synthesized D,L-pantolactone. Such a process, however, has disadvantages in that it requires the use of expensive optical resolving agents such as quinine or brucine and further that the recovery of D-pantolactone is not easy. In order to solve such problems, the present inventors already proposed an optical resolving method by an enzymatic asymmetric hydrolysis of D,L-pantolactone in Unexamined Japanese Patent Publication (KOKAI TOKKYO) Nos. Hei 03-65,198 and Hei 04-144,681.
Thus, it is a process for the production of D-pantolactone, wherein the D-pantolactone in D,L-pantolactone mixtures is selectively subjected to an asymmetric hydrolysis using a microorganism possessing a lactone-hydrolyzing activity to form D-pantoic acid, which is then separated and converted into D-pantolactone, wherein said microorganism is a member selected from the group consisting of microorganisms belonging to the genera: Fusarium, Cylindrocarpon, Gibberella, Aspergillus, Penicillium, Rhizopus, Volutella, Gliocladium, Eurotium, Nectoria, Schizophyllum, Myrothecium, Neurospora, Acremonium, Tuberculina, Absidia, Sporothrix, Verticillium and Arthroderma. It is also a process for producing D-pantolactone hydrolase which comprises using a microorganism belonging to the above-mentioned genus.
However, it cannot be always said that many of those microorganisms disclosed as above possess a hydrolyzing activity to such an extent that they are immediately applicable in industry. Furthermore, in increasing the enzymatic activity of said microorganisms to an industrially applicable level, troublesome and difficult investigations requiring long time are needed for establishing conditions for growth of cells, conditions for enzyme activity induction, etc. There is another problem that, since said microorganisms are true fungus, their cell bodies are in variously shaped hyphae and, as compared with bacteria having a single-shape, it is considerably difficult to prepare immobilized cells which are advantageous for industrial production. There is still another problem that, in purifying the enzyme from the cells, its recovery rate is considerably poor so far as D-pantolactone hydrolase is concerned.
An object of the present invention is to solve those problems and also to provide means for making a significant increase of the enzymatic activity possible, for example, means for modifying and improving the D-pantolactone hydrolase per se.
Thus, one aspect of the present invention is to disclose and provide a novel gene which codes for a protein having either a naturally-occurring D-pantolactone hydrolase activity (such as a Fusarium oxysporum D-pantolactone hydrolase activity) or an activity substantially equivalent thereto; a host cell transformed with DNA containing a nucleotide sequence coding for said protein; a process for producing said protein via using said host cell; and uses of said proteins and host cells.
The present invention directed to a gene coding for D-pantolactone hydrolase isolated from the above-mentioned microorganisms possessing the ability to hydrolyze a lactone and a system, with a high efficiency and rich productivity, for producing D-pantolactone is successfully developed through utilizing the D-pantolactone hydrolase gene isolated as such, not only solves the above-mentioned various problems but also greatly contributes to the development of enzymes possessing the ability to hydrolyze a lactone, together with new functions; and to the development of techniques using the novel enzyme. Particularly, the present inventors have succeeded in isolating a novel gene coding for a hydrolase with a D-pantolactone hydrolyzing ability, derived from microorganisms of the genus Fusarium (such as Fusarium oxysporum) which produces the D-pantolactone hydrolase, whereby the present invention has been achieved.
The present invention relates to:
(i) a protein having a natural D-pantolactone hydrolase activity or an activity substantially equivalent thereto or a salt thereof; or
(ii) a protein having a primary structural conformation substantially equivalent thereto or a salt thereof;
(iii) a characteristic partial peptide of said protein or a salt thereof;
(iv) genes, such as DNA and RNA, coding for said protein;
(v) vectors or plasmids, containing said gene operably in a gene recombination technique;
(vi) host cells transformed with such a vector, etc.;
(vii) a process for producing said protein or a salt thereof which comprises culturing said host cell;
(viii) a process for producing D-pantolactone which comprises an optical resolution of D,L-pantolactone with such a gene-manipulated host cell (transformant), such a recombinant protein or a salt thereof, etc.; and
(ix) a system means, such as an immobilized enzyme, for producing D-pantolactone.
In the present invention, a preferred recombinant protein is a D-pantolactone hydrolase having an amino acid sequence of SEQ ID NO:1 or an amino acid sequence substantially equivalent thereto, or a salt thereof.
Accordingly, one aspect of the present invention is:
(1) a protein having a naturally-occurring D-pantolactone hydrolase activity or an activity substantially equivalent thereto or having a primary structural conformation substantially equivalent thereto, or a salt thereof;
(2) the protein according to the above (1), wherein said protein having a naturally-occurring D-pantolactone hydrolase activity is originating in a microorganism belonging to a member selected from the group consisting of genera: Fusarium, Cylindrocarpon, Gibberella, Aspergillus, Penicillium, Rhizopus, Volutella, Gliocladium, Eurotium, Nectoria, Schizophyllum, Myrothecium, Neurospora, Acremonium, Tuberculina, Absidia, Sporothrix, Verticillium and Arthroderma;
(3) the protein according to the above (1), wherein said protein having a naturally-occurring D-pantolactone hydrolase activity is originating in the genus Fusarium;
(4) the protein according to any of the above (1) to (3), which is a D-pantolactone hydrolase, or a salt thereof, having an amino acid sequence represented by SEQ ID NO:1 or an amino acid sequence substantially equivalent thereto;
(5) the protein according to any of the above (1) to (4), which is produced by expressing an exogenous DNA sequence in procaryotic host cells;
(6) the protein according to any of the above (1) to (5), which has an amino acid sequence represented by SEQ ID NO:1 or the substantially same amino acid sequence as it has;
(7) a partial peptide, or a salt thereof, of the protein according to any of the above (1) to (6);
(8) a nucleic acid having a nucleotide sequence coding for the protein or partial peptide thereof according to any of the above (1) to (7);
(9) the nucleic acid according to the above (8), which has a nucleotide sequence having a portion corresponding to an open reading frame in the nucleotide sequence of SEQ ID NO:2 or a nucleotide sequence having an activity substantially equivalent thereto;
(10) a vector carrying the nucleic acid according to the above (8) or (9);
(11) a transformant wherein the vector according to the above (10) is harbored;
(12) a process for producing the protein or partial peptide thereof according to any of the above (1) to (7), including a D-pantolactone hydrolase or a salt thereof, which comprises:
culturing the transformant according to the above (11) in a nutrient medium suitable for growing said transformant to produce, as a recombinant protein, the protein or partial peptide thereof according to any of the above (1) to (7), including said D-pantolactone hydrolase or a salt thereof; and
(13) a process for producing D-pantolactone, which comprises:
carrying out an optical resolution of D,L-pantolactone in the presence of
(i) the protein or partial peptide thereof according to any of the above (1) to (7) or
(ii) the transformant according to the above (11).
More specifically, the present invention provides a D-pantolactone hydrolase, or a salt thereof, having an amino acid sequence of SEQ ID NO:1.